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Inhibitors that stabilize a closed RAF kinase domain conformation induce dimerization

Abstract

RAF kinases have a prominent role in cancer. Their mode of activation is complex but critically requires dimerization of their kinase domains. Unexpectedly, several ATP-competitive RAF inhibitors were recently found to promote dimerization and transactivation of RAF kinases in a RAS-dependent manner and, as a result, undesirably stimulate RAS/ERK pathway–mediated cell growth. The mechanism by which these inhibitors induce RAF kinase domain dimerization remains unclear. Here we describe bioluminescence resonance energy transfer–based biosensors for the extended RAF family that enable the detection of RAF dimerization in living cells. Notably, we demonstrate the utility of these tools for profiling kinase inhibitors that selectively modulate RAF dimerization and for probing structural determinants of RAF dimerization in vivo. Our findings, which seem generalizable to other kinase families allosterically regulated by kinase domain dimerization, suggest a model whereby ATP-competitive inhibitors mediate RAF dimerization by stabilizing a rigid closed conformation of the kinase domain.

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Figure 1: Development of BRET-based RAF dimerization biosensors.
Figure 2: Profiling RAF inhibitors using RAF dimerization biosensors.
Figure 3: Development of a RAS-dependent CRAF-BRAF dimerization biosensor.
Figure 4: A high-throughput chemical screen using the CRAFKD–BRAFKD biosensor identifies new modulators of RAF dimerization.
Figure 5: Screening of a kinase inhibitor library reveals widespread off-target effects on RAF dimerization.
Figure 6: Probing the binding mode of RAF dimer inducers with BRAF mutant biosensors.

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References

  1. Roberts, P.J. & Der, C.J. Targeting the Raf-MEK-ERK mitogen-activated protein kinase cascade for the treatment of cancer. Oncogene 26, 3291–3310 (2007).

    Article  CAS  PubMed  Google Scholar 

  2. Wellbrock, C., Karasarides, M. & Marais, R. The RAF proteins take centre stage. Nat. Rev. Mol. Cell Biol. 5, 875–885 (2004).

    Article  CAS  PubMed  Google Scholar 

  3. Dhomen, N. & Marais, R. New insight into BRAF mutations in cancer. Curr. Opin. Genet. Dev. 17, 31–39 (2007).

    Article  CAS  PubMed  Google Scholar 

  4. Schubbert, S., Shannon, K. & Bollag, G. Hyperactive Ras in developmental disorders and cancer. Nat. Rev. Cancer 7, 295–308 (2007).

    Article  CAS  PubMed  Google Scholar 

  5. Clapéron, A. & Therrien, M. KSR and CNK: two scaffolds regulating RAS-mediated RAF activation. Oncogene 26, 3143–3158 (2007).

    Article  PubMed  Google Scholar 

  6. Garnett, M.J., Rana, S., Paterson, H., Barford, D. & Marais, R. Wild-type and mutant B-RAF activate C-RAF through distinct mechanisms involving heterodimerization. Mol. Cell 20, 963–969 (2005).

    Article  CAS  PubMed  Google Scholar 

  7. Rajakulendran, T., Sahmi, M., Lefrancois, M., Sicheri, F. & Therrien, M. A dimerization-dependent mechanism drives RAF catalytic activation. Nature 461, 542–545 (2009).

    Article  CAS  PubMed  Google Scholar 

  8. Rushworth, L.K., Hindley, A.D., O'Neill, E. & Kolch, W. Regulation and role of Raf-1/B-Raf heterodimerization. Mol. Cell Biol. 26, 2262–2272 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Weber, C.K., Slupsky, J.R., Kalmes, H.A. & Rapp, U.R. Active Ras induces heterodimerization of cRaf and BRaf. Cancer Res. 61, 3595–3598 (2001).

    CAS  PubMed  Google Scholar 

  10. Halilovic, E. & Solit, D.B. Therapeutic strategies for inhibiting oncogenic BRAF signaling. Curr. Opin. Pharmacol. 8, 419–426 (2008).

    Article  CAS  PubMed  Google Scholar 

  11. Bollag, G. et al. Clinical efficacy of a RAF inhibitor needs broad target blockade in BRAF-mutant melanoma. Nature 467, 596–599 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Joseph, E.W. et al. The RAF inhibitor PLX4032 inhibits ERK signaling and tumor cell proliferation in a V600E BRAF-selective manner. Proc. Natl. Acad. Sci. USA 107, 14903–14908 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Hatzivassiliou, G. et al. RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth. Nature 464, 431–435 (2010).

    Article  CAS  PubMed  Google Scholar 

  14. Heidorn, S.J. et al. Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF. Cell 140, 209–221 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Poulikakos, P.I., Zhang, C., Bollag, G., Shokat, K.M. & Rosen, N. RAF inhibitors transactivate RAF dimers and ERK signalling in cells with wild-type BRAF. Nature 464, 427–430 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Poulikakos, P.I. & Rosen, N. Mutant BRAF melanomas—dependence and resistance. Cancer Cell 19, 11–15 (2011).

    Article  CAS  PubMed  Google Scholar 

  17. Poulikakos, P.I. et al. RAF inhibitor resistance is mediated by dimerization of aberrantly spliced BRAF(V600E). Nature 480, 387–390 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Solit, D.B. & Rosen, N. Resistance to BRAF inhibition in melanomas. N. Engl. J. Med. 364, 772–774 (2011).

    Article  CAS  PubMed  Google Scholar 

  19. Bacart, J., Corbel, C., Jockers, R., Bach, S. & Couturier, C. The BRET technology and its application to screening assays. Biotechnol. J. 3, 311–324 (2008).

    Article  CAS  PubMed  Google Scholar 

  20. Breton, B. et al. Multiplexing of multicolor bioluminescence resonance energy transfer. Biophys. J. 99, 4037–4046 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Kocan, M., See, H.B., Seeber, R.M., Eidne, K.A. & Pfleger, K.D. Demonstration of improvements to the bioluminescence resonance energy transfer (BRET) technology for the monitoring of G protein–coupled receptors in live cells. J. Biomol. Screen. 13, 888–898 (2008).

    Article  CAS  PubMed  Google Scholar 

  22. James, J.R., Oliveira, M.I., Carmo, A.M., Iaboni, A. & Davis, S.J. A rigorous experimental framework for detecting protein oligomerization using bioluminescence resonance energy transfer. Nat. Methods 3, 1001–1006 (2006).

    Article  CAS  PubMed  Google Scholar 

  23. Röring, M. et al. Distinct requirement for an intact dimer interface in wild-type, V600E and kinase-dead B-Raf signalling. EMBO J. 31, 2629–2647 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  24. Ritt, D.A., Monson, D.M., Specht, S.I. & Morrison, D.K. Impact of feedback phosphorylation and Raf heterodimerization on normal and mutant B-Raf signaling. Mol. Cell Biol. 30, 806–819 (2010).

    Article  CAS  PubMed  Google Scholar 

  25. Dar, A.C. & Shokat, K.M. The evolution of protein kinase inhibitors from antagonists to agonists of cellular signaling. Annu. Rev. Biochem. 80, 769–795 (2011).

    Article  CAS  PubMed  Google Scholar 

  26. Whittaker, S. et al. Gatekeeper mutations mediate resistance to BRAF-targeted therapies. Sci. Transl. Med. 2, 35ra41 (2010).

    Article  PubMed  Google Scholar 

  27. Zhang, J.H., Chung, T.D. & Oldenburg, K.R. A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J. Biomol. Screen. 4, 67–73 (1999).

    Article  CAS  PubMed  Google Scholar 

  28. Tsai, J. et al. Discovery of a selective inhibitor of oncogenic B-Raf kinase with potent antimelanoma activity. Proc. Natl. Acad. Sci. USA 105, 3041–3046 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Wan, P.T. et al. Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF. Cell 116, 855–867 (2004).

    Article  CAS  PubMed  Google Scholar 

  30. Lee, J.C. et al. A protein kinase involved in the regulation of inflammatory cytokine biosynthesis. Nature 372, 739–746 (1994).

    Article  CAS  PubMed  Google Scholar 

  31. Hall-Jackson, C.A., Goedert, M., Hedge, P. & Cohen, P. Effect of SB 203580 on the activity of c-Raf in vitro and in vivo. Oncogene 18, 2047–2054 (1999).

    Article  CAS  PubMed  Google Scholar 

  32. Kalmes, A., Deou, J., Clowes, A.W. & Daum, G. Raf-1 is activated by the p38 mitogen-activated protein kinase inhibitor, SB203580. FEBS Lett. 444, 71–74 (1999).

    Article  CAS  PubMed  Google Scholar 

  33. McKay, M.M., Ritt, D.A. & Morrison, D.K. RAF Inhibitor-Induced KSR1/B-RAF binding and its effects on ERK cascade signaling. Curr. Biol. 21, 563–568 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. King, A.J. et al. Demonstration of a genetic therapeutic index for tumors expressing oncogenic BRAF by the kinase inhibitor SB-590885. Cancer Res. 66, 11100–11105 (2006).

    Article  CAS  PubMed  Google Scholar 

  35. Wang, Z. et al. Structural basis of inhibitor selectivity in MAP kinases. Structure 6, 1117–1128 (1998).

    Article  CAS  PubMed  Google Scholar 

  36. Anastassiadis, T., Deacon, S.W., Devarajan, K., Ma, H. & Peterson, J.R. Comprehensive assay of kinase catalytic activity reveals features of kinase inhibitor selectivity. Nat. Biotechnol. 29, 1039–1045 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Packer, L.M. et al. Nilotinib and MEK inhibitors induce synthetic lethality through paradoxical activation of RAF in drug-resistant chronic myeloid leukemia. Cancer Cell 20, 715–727 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Barrios-Rodiles, M. et al. High-throughput mapping of a dynamic signaling network in mammalian cells. Science 307, 1621–1625 (2005).

    Article  CAS  PubMed  Google Scholar 

  39. Jeffrey, P.D. et al. Mechanism of CDK activation revealed by the structure of a cyclinA–CDK2 complex. Nature 376, 313–320 (1995).

    Article  CAS  PubMed  Google Scholar 

  40. Sicheri, F. & Kuriyan, J. Structures of Src-family tyrosine kinases. Curr. Opin. Struct. Biol. 7, 777–785 (1997).

    Article  CAS  PubMed  Google Scholar 

  41. Kornev, A.P., Haste, N.M., Taylor, S.S. & Eyck, L.F. Surface comparison of active and inactive protein kinases identifies a conserved activation mechanism. Proc. Natl. Acad. Sci. USA 103, 17783–17788 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Kornev, A.P. & Taylor, S.S. Defining the conserved internal architecture of a protein kinase. Biochim. Biophys. Acta 1804, 440–444 (2010).

    Article  CAS  PubMed  Google Scholar 

  43. Bukhtiyarova, M., Karpusas, M., Northrop, K., Namboodiri, H.V. & Springman, E.B. Mutagenesis of p38α MAP kinase establishes key roles of Phe169 in function and structural dynamics and reveals a novel DFG-OUT state. Biochemistry 46, 5687–5696 (2007).

    Article  CAS  PubMed  Google Scholar 

  44. Axten, J.M. et al. Discovery of 7-methyl-5-(1-{[3-(trifluoromethyl)phenyl]acetyl}-2,3-dihydro-1H-indol-5-yl)-7H-p yrrolo[2,3-d]pyrimidin-4-amine (GSK2606414), a potent and selective first-in-class inhibitor of protein kinase r (PKR)-like endoplasmic reticulum kinase (PERK). J. Med. Chem. 55, 7193–7207 (2012).

    Article  CAS  PubMed  Google Scholar 

  45. Dey, M. et al. Mechanistic link between PKR dimerization, autophosphorylation, and eIF2α substrate recognition. Cell 122, 901–913 (2005).

    Article  CAS  PubMed  Google Scholar 

  46. Taylor, S.S., Haste, N.M. & Ghosh, G. PKR and eIF2α: integration of kinase dimerization, activation, and substrate docking. Cell 122, 823–825 (2005).

    Article  CAS  PubMed  Google Scholar 

  47. Korennykh, A.V. et al. The unfolded protein response signals through high-order assembly of Ire1. Nature 457, 687–693 (2009).

    Article  CAS  PubMed  Google Scholar 

  48. Sen, B. et al. Kinase-impaired BRAF mutations in lung cancer confer sensitivity to dasatinib. Sci. Transl. Med. 4, 136ra170 (2012).

    Article  Google Scholar 

  49. Dar, A.C., Dever, T.E. & Sicheri, F. Higher-order substrate recognition of eIF2α by the RNA-dependent protein kinase PKR. Cell 122, 887–900 (2005).

    Article  CAS  PubMed  Google Scholar 

  50. Boussif, O. et al. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc. Natl. Acad. Sci. USA 92, 7297–7301 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Adams, P.D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Ciruela, F. Fluorescence-based methods in the study of protein-protein interactions in living cells. Curr. Opin. Biotechnol. 19, 338–343 (2008).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank B. Breton and M. Audet for advice with the BRET2 system, D. Uehling and co-workers at the Ontario Institute for Cancer Research for GSK2606414, and the Institut de recherche en immunologie et en cancérologie (IRIC) high-throughput screening platform. IRIC is supported by the Canadian Center of Excellence in Commercialization and Research, the Canada Foundation for Innovation and by the Fonds de Recherche du Québec en Santé. H.L. is a recipient of Cancer Research Society and Canadian Institutes for Health Research (CIHR) Banting postdoctoral fellowships. N.T. is a recipient of the CIHR Canadian Graduate Scholarship. M.B., F.S. and M.T. hold Canada Research Chairs. This work was supported by operating funds from the Canadian Cancer Society to M.T. (018046) and from the CIHR to M.T. (MOP119443) and F.S. (MOP36399).

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H.L., N.T., F.S. and M.T. designed the experiments and wrote the manuscript. M.B. contributed to the theoretical framework surrounding BRET assay development and participated in the analysis of the BRET data and in the revision of the manuscript. H.L., with assistance from G.G., A.P., S.G. and J.D., conducted cell-based BRET analyses, the high-throughput chemical screen and TR-FRET in vitro binding assays. N.T., with assistance from D.Y.L.M., J.J.L. and H.L., performed AUC and X-ray structure analyses.

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Correspondence to Frank Sicheri or Marc Therrien.

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Competing interests

H.L., F.S. and M.T. have filed a patent covering the BRET-based method described in this study for detecting RAF dimerization.

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Lavoie, H., Thevakumaran, N., Gavory, G. et al. Inhibitors that stabilize a closed RAF kinase domain conformation induce dimerization. Nat Chem Biol 9, 428–436 (2013). https://doi.org/10.1038/nchembio.1257

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